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WO2014074453A1 - Compositions et procédés pour l'oxygénation d'acides nucléiques contenant de la 5-méthylpyrimidine - Google Patents

Compositions et procédés pour l'oxygénation d'acides nucléiques contenant de la 5-méthylpyrimidine Download PDF

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WO2014074453A1
WO2014074453A1 PCT/US2013/068298 US2013068298W WO2014074453A1 WO 2014074453 A1 WO2014074453 A1 WO 2014074453A1 US 2013068298 W US2013068298 W US 2013068298W WO 2014074453 A1 WO2014074453 A1 WO 2014074453A1
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Prior art keywords
hmc
dna
buffer
identity
composition according
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Inventor
Yu Zheng
Lana SALEH
June PAIS
Nan Dai
Richard J. Roberts
Ivan R. CORREA
Megumu MABUCHI
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New England Biolabs Inc
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New England Biolabs Inc
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Definitions

  • 5-methylcytosine (5-mC) has been linked to gene expression and its distribution in the genome plays an important role in epigenetics.
  • 5-mC 5-methylcytosine
  • 5-hmC 5- hydroxymethylcytosine
  • Three enzymes named TET1/2/3 have been shown in human and mouse to be responsible for oxidizing 5-mC to 5-hmC.
  • TET enzymes belong to the broad family of Fe(II)/2-oxo-glutarate-dependent (20GFE) oxygenases, which use 2-oxo-glutarate (20G), as co-substrate, and ferrous ion (Fe(II)) as cofactor.
  • 2-oxo-glutarate (20G) as co-substrate
  • Fe(II) ferrous ion
  • oxidation products identified as 5-hmC, 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC).
  • 5-caC is believed to be excised via the action of DNA glycosylases and replaced by the unmodified cytosine.
  • TET enzymes are very large proteins and hence it has been problematic to make these proteins in recombinant form and in sufficient quantities to use as a research reagent.
  • Neigleria gruberi has not been previously reported to contain 5-mC or 5-hmC, the present inventors have surprisingly discovered that a protein from N. gruberi can be used in vitro to convert 5-mC to oxidized cytosines. That protein can be purified from natural sources or produced recombinantly, optionally as a fusion protein with another amino acid sequence to facilitate its purification or use.
  • a fusion protein in which a binding domain is fused to a recombinant 5-methylpyrimidine oxygenase (mYOXl) having a size less than 600 amino acids and having a catalytic domain having 90% or 100% identity with the amino acid sequence of SEQ ID NO: l.
  • mYOXl 5-methylpyrimidine oxygenase
  • the mYOXl has an amino acid sequence with at least 90% identity (or more, such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity) to amino acids 209-296, 160-297, 154- 304 or 1-321 of the amino acid sequence of SEQ ID NO: 2 (mYOXl), and/or with the corresponding amino acids of any one of SEQ ID NOs: 3-9 as aligned with SEQ ID NO: 2 in Figure 2B, optionally while retaining 90% or 100% identity with the amino acid sequence of SEQ ID NO: l.
  • identity or more, such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity
  • the mYOXl has an amino acid sequence with at least 90% identity (or more, such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity) to the entire length of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 9.
  • the binding domain is capable of recognizing and binding to another molecule.
  • the binding domain is a histidine tag ("His-tag”), a maltose-binding protein, a chitin-binding domain, or a DNA-binding domain, which may include a zinc finger and/or a transcription activator-like (TAL) effector domain.
  • the fusion protein can be used as a mYOXl (such as a 5-mC oxygenase or a thymine hydroxylase) in single-or double-stranded DNA or in RNA, typically at a pH of about 6 (generally between 5.5 and 6.5) to about 8, and, in some embodiments, at a pH of about 6 to about pH 7.5.
  • embodiments provide buffered compositions containing a purified mYOXl having a size less than 600 amino acids and having a catalytic domain having 90% or 100% identity with the amino acid sequence of SEQ ID NO: l.
  • the mYOXl has an amino acid sequence with at least 90% identity (or more, such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity) to amino acids 209-296, 160-297, 154-304 or 1-321 of the amino acid sequence of SEQ ID NO:2, and/or with the corresponding amino acids of any one of SEQ ID NOs:3-9 as aligned with SEQ ID NO: 2 in Figure 2B, optionally while retaining 90% or 100% identity with the amino acid sequence of SEQ ID NO: l.
  • the mYOXl has an amino acid sequence with at least 90% identity (or more, such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity) to the entire length of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, or 9.
  • the amino acid sequence with at least 90% identity (or more, such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity) to the entire length of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, or 9.
  • composition contains glycerol; and/or contains Fe(II), as cofactor, and a- ketoglutarate, as co-substrate, for the enzyme.
  • the composition does not contain ATP, which can interfere with subsequent oxidation of hydroxy methylated nucleotides; in other embodiments, the composition does contain ATP (e.g. to inhibit further oxidation).
  • the composition is optionally at a pH from about 6 to about 8. In certain embodiments, the pH is about 6, or is from about 6 to about 7.5.
  • the buffered compositions can be used to generate a variety of oxidation products of 5-mC, including 5-hmC, 5-fC, and 5-caC. The distribution of oxidation products can be varied by varying the pH of the reaction buffer.
  • the pH of the buffered composition is about 6; about 6.0 to about 6.5; about 6.0 to about 7.0; about 6.0 to about 7.5; about 6.0 to about 8.0; about 6.5 to about 7.0; about 6.5 to about 7.5; about 6.5 to about 8.0; about 7.0 to about 8.0; or about 7.5 to about 8.0.
  • the buffered compositions also include a nucleic acid, such as single- or double-stranded DNA that may include 5-mC (as a substrate for the enzyme) and/or one or more of 5-hmC, 5-fC, or 5- caC (naturally-occurring, and/or resulting from the activity of the enzyme).
  • a nucleic acid such as single- or double-stranded DNA that may include 5-mC (as a substrate for the enzyme) and/or one or more of 5-hmC, 5-fC, or 5- caC (naturally-occurring, and/or resulting from the activity of the enzyme).
  • kits for modifying nucleic acids include a purified mYOXl having a size less than 600 amino acids and having a catalytic domain having 90% or 100% identity with the amino acid sequence of SEQ ID NO: 1, or any one of the buffered compositions or fusion proteins described above, together with a separate reaction buffer.
  • the mYOXl has an amino acid sequence with at least 90% identity (or more, such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity) to amino acids 209-296, 160-297, 154-304 or 1-321 of the amino acid sequence of SEQ ID NO : 2, optionally while retaining 90% or 100% identity with the amino acid sequence of SEQ ID NO : l .
  • the reaction buffer has a pH typically from about 6 to about 8, and may contain contains Fe(II) and/or a-ketoglutarate.
  • the pH of the reaction buffer is about 6; about 6.0 to about 6.5; about 6.0 to about 7.0; about 6.0 to about 7.5; about 6.0 to about 8.0; about 6.5 to about 7.0; about 6.5 to about 7.5; about 6.5 to about 8.0; about 7.0 to about 8.0; or about 7.5 to about 8.0.
  • the kit may also include a nucleic acid such as single- or double- stranded DNA that may include one or more 5-mC residues. Also, or alternatively, the kit may include: a reducing agent, such as sodium
  • borohydride or an additive, such as cobalt chloride; a ⁇ -glycosyltransferase (BGT) and UDP-glucose and/or UDP-glucosamine; a DNA glycosylase such as thymine DNA glycosylase; and/or an endonuclease, such as an endonuclease that cleaves DNA containing 5-hmC more efficiently than it cleaves DNA containing p-glucosyl-oxy-5-methylcytosine (5-ghmC) (e.g. AbaSI).
  • BGT ⁇ -glycosyltransferase
  • UDP-glucose and/or UDP-glucosamine UDP-glucose and/or UDP-glucosamine
  • a DNA glycosylase such as thymine DNA glycosylase
  • an endonuclease such as an endonuclease that cleaves DNA containing 5-hmC more efficiently than it
  • Embodiments also provide kits for detecting the 5-mC in double- stranded or single-stranded DNA or RNA by sequencing, e.g., single- molecular sequencing such as Pacific Biosciences platform.
  • the kits include a purified mYOXl having a size less than 600 amino acids and having a catalytic domain having 90% or 100% identity with the amino acid sequence of SEQ ID NO: l, or any one of the buffered compositions or fusion proteins described above, together with a separate reaction buffer.
  • the mYOXl has an amino acid sequence with at least 90% identity (or more, such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity) to amino acids 209-296, 160-297, 154-304 or 1-321 of the amino acid sequence of SEQ ID NO: 2, optionally while retaining 90% or 100% identity with the amino acid sequence of SEQ ID NO: l.
  • the reaction buffer has a pH typically from about 6 to about 8, and may contain contains Fe(II) and/or a-ketoglutarate.
  • the pH of the reaction buffer is about 6; about 6.0 to about 6.5; about 6.0 to about 7.0; about 6.0 to about 7.5; about 6.0 to about 8.0; about 6.5 to about 7.0; about 6.5 to about 7.5; about 6.5 to about 8.0; about 7.0 to about 8.0; or about 7.5 to about 8.0.
  • the kit may contain other DNA/RNA repair enzymes for the DNA or RNA to be used in the sequencing platforms.
  • embodiments provide methods for differentiating a
  • the method includes: reacting the isolated genome or genome fragment containing 5-mC and 5-hmC with UDP-glucose or UDP-glucosamine, a glycosy transferase for transferring glucose or glucosamine to the 5-hmC, and one of the previously described fusion proteins or buffered compositions; cleaving the glucosylated template with a modification-dependent
  • the method includes: reacting the isolated genome or genome fragment containing 5-mC and 5-hmC with UDP-glucosamine and a glycosy transferase for transferring glucosamine to the 5-hmC; subsequently reacting the isolated genome or genome fragment with one of the previously described fusion proteins or buffered compositions and optionally with a reducing agent; cleaving the template with a modification-dependent endonuclease that is capable of selectively cleaving a 5-hmC and not a 5- ghmC; and differentiating the 5-mC from one or more of its oxidation products by an altered cleavage pattern.
  • the modification-dependent endonuclease is optionally AbaSI.
  • Embodiments also provide methods of modifying a 5-mC oxygenase by introducing random or targeted mutations and changing the specificity of the enzyme so as to exclusively oxidize 5-mC to 5-hmC.
  • Figure 1 shows a phylogram of mYOXl in Naegleria gruberi and TET proteins based on the ClustalW multiple sequence alignment.
  • TETl_hs_C human TET1 truncated C-terminus
  • TETl_mm_C mouse TET1 truncated C- terminus
  • TET2_hs_C human TET2 truncated C-terminus
  • TET2_mm_C mouse TET2 truncated C-terminus
  • TET3_hs_C human TET3 truncated C- terminus
  • TET3_mm_C mouse TET3 truncated C-terminus.
  • Figure 2A-B shows eight mYOX proteins in Naegleria gruberi and their alignments. This family of problems has a consensus sequence
  • Figure 2A shows the conserved domain structure of the 8 mYOX proteins anchored by the 2OGFE catalytic domain. An additional domain, a CHROMO domain, was detected in one of the proteins.
  • Figure 2B shows multiple sequence alignment of the 2OGFE catalytic domain sequences in mYOX proteins. Alignment was performed by the PROMALS program (http://prodata.swmed.edu/promals/promals.php).
  • Figure 3 shows a single band of purified recombinant mYOXl having a molecular weight of 37,321 Dalton on an SDS-PAGE.
  • Figure 4A-C shows the activity of mYOXl.
  • Figure 4A shows the activity on double-stranded DNA with 24 fully-methylated CpG sites ("24x oligo").
  • Figure 4B shows the activity on plasmid DNA ("pTXBl-M.Sssl").
  • Figure 4C shows the activity on genomic DNA ("IMR90"). All substrate DNA contained 5-mC. The generation of 5-hmC, 5-fC and
  • 5-caC was monitored by liquid chromatography. The generation of 5-hmC was dependent on mYOXl, since no 5-hmC was detected in the absence of the enzyme. In addition, mYOXl was able to convert thymine to 5-hmU, 5- fU and 5-caU (data not shown). These results indicate that mYOXl is an active 5-mC oxygenase and thymine hydroxylase.
  • Figure 5 shows methods for mapping methylome
  • mYOXs or, more specifically, 5-mC oxygenases that can use 2OG, as co-substrate, and ferrous ion (Fe(II)), as cofactor.
  • This novel family whose members are referred to in this application as mYOXs, is distantly related to the TET proteins, as shown in the phylogram of Figure 1, sharing about 15%
  • mYOXs Compared to TET proteins, mYOXs have several advantages as reagents for oxygenating 5-mC. With sizes in the range of 174-583aa, mYOXs are substantially smaller than enzymes of the TET family (which are ⁇ 1600-2000aa), facilitating their recombinant production. Their small size renders these enzymes suitable as components in fusion proteins with, for example, DNA binding domains such as zinc fingers, and/or one or more additional enzymatic domains such as a glycosylase to promote the eventual excision of the modified cytosine.
  • mYOXs operate more efficiently at pH 7.5 or less (e.g. at about pH 6), and do not require ATP which is significant because it reduces the possibility of side reactions, for example,
  • mYOXl over TET proteins as research reagents includes its improved catalytic efficiency. For example, stoichiometrically fewer enzyme molecules are needed to oxidize 5-mCs when using mYOXl rather than a TET enzyme.
  • mYOXs can be cloned and purified from Naegleria gruberi, a free-living single-cell protist as described in Example 1. Host cells suitable for
  • expression include E. coli, yeast and insect cell systems producing greater than 10 pg/l, 20 pg/l, 30 pg/l, 50 pg/l, 70 pg/l, 100 pg/l, 200 pg/l, 300 pg/l, 400 ⁇ g/l, 500 g/l and as much as 10 mg/liter of culture.
  • a unit amount of mYOXl is able to convert lpmol of 5-mC on DNA in 30 minutes at 34°C in IX mYOXl reaction buffer at pH 6.0 (unit definition).
  • mYOX protein sequences are provided in the following table:
  • Figure 2A-B depicts the common structure among these 8 mYOX proteins, including a conserved domain structure 9 (see panel A) and conserved sequences in that conserved domain as revealed by a multiple sequence alignment (see panel B).
  • These 8 proteins share a common consensus sequence: (R/K)X 4 HXDXi 2 GXi 8 - 3 oDXioHXVX 7 - 72 RX 5 FA (SEQ ID NO: l).
  • Biochemical assays for characterization of these enzymes includes: non-quantitative assays, e.g., dot-blot assay using product-specific
  • mYOX enzymes may oxidize 5-mC through intermediate product forms to 5-caC. Mutants of these enzymes can be assayed for significant bias toward one oxidized form over another for example, a significant bias for conversion of 5-mC to 5-hmC or 5-mC to 5-fC or 5-caC. This allows direct detection of a single oxidation form and also a temporal means of tracking change in the oxidation state of modified nucleotides in the genome and correlation of these states and their changes to phenotypic change.
  • Additional mutants may include those that only oxidize 5-mC, or 5- hmC, or 5-fC, but not other modified forms of cytosine.
  • a mutant may oxidize 5-hmC to 5-fC or 5-caC, but will not work on 5-mC. These mutants may enable a variety of in vitro epigenomic mapping techniques.
  • Mutants can be engineered using standard techniques such as rational design by site-directed mutagenesis based on enzyme 3D structures and screening/selection methods in large random mutant libraries.
  • Embodiments of the invention include uses of mYOXs for mapping of both methylome and hydroxymethylome.
  • differentiation processes in eukaryotic organisms can be studied using N. gruberi as a model system.
  • N. gruberi is a single-cell protist that can differentiate from an ameoba form to a flagella form in a synchronous manner. It thus forms a model system to study dynamic methylome/hydroxymethylome changes that contribute to the gene/pathway regulation during differentiation.
  • the 5-mC in the genomic DNA can be converted to 5-hmC using an mYOX such as mYOXl or other member of the mYOX family.
  • Reducing agents such as NaBH4, can be used in the reaction to ensure that any oxidation products in the form of 5-fC or 5-caC or naturally occurring instances of the same are converted to 5-hmC.
  • Any chemical or enzyme capable of promoting the reduction of 5-fC or 5-caC to 5-hmC can be used for that purpose.
  • Many water-soluble metal or metalloid hydrides are able to reduce aldehydes and/or carboxylic acids to alcohols. Examples of such reducing agents are sodium borohydride and related compounds where from 1 to 3 of the hydrogens are replaced by other moieties, such as cyano and alkoxy containing up to about 5 carbon atoms.
  • substituted borohydrides include cyanoborohydride, dicyanoborohydride, methoxyborohydride, dimethoxyborohydride, trimethoxyborohydride, ethoxyborohydride, diethoxyborohydride, triethoxyborohydride,
  • butoxyborohydride dibutoxyborohydride, tributoxyborohydride, and so forth.
  • water-soluble metal hydrides include lithium borohydride, potassium borohydride, zinc borohydride, aluminum borohydride, zirconium borohydride, beryllium borohydride, and sodium bis(2- methoxyethoxy)aluminium hydride.
  • Sodium borohydride can also be used in combination with a metal halide, such as cobalt(II), nickel(II), copper(II), zinc(II), cadmium (II), calcium (II), magnesium(II), aluminum(III), titanium (IV), hafnium(IV), or rhodium(III), each of which can be provided as a chloride, bromide, iodide, or fluoride salt.
  • sodium borohydride can be used in combination with iodine, bromine, boron trifluoride diethyl etherate, trifluoroacetic acid, catechol-trifluoroacetic acid, sulfuric acid, or diglyme.
  • Particular reducing strategies include the combination of potassium borohydride with lithium chloride, zinc chloride, magnesium chloride, or hafnium chloride; or the combination of lithium borohydride and
  • chlorotrimethylsilane Other reducing strategies include the use of borane, borane dimethyl sulfide complex, borane tetrahydrofuran complex, borane- ammonia complex, borane morpholine complex, borane dimethylamine complex, borane trimethylamine complex, borane N,N-diisopropylethylamine complex, borane pyridine complex, 2-picoline borane complex, borane 4- methylmorpholine complex, borane tert-butylamine complex, borane
  • triphenylphosphine complex borane ⁇ , ⁇ -diethylaniline complex, borane di(tert-butyl)phosphine complex, borane diphenylphosphine complex, borane ethylenediamine complex, or lithium ammonia borane.
  • Alternative reducing strategies include the reduction of carboxylic acids via the formation of hydroxybenzotriazole esters, carboxy methyleniminium chlorides, carbonates, O-acylisoureas, acyl fluorides, cyanurates, mixed anhydrides, arylboronic anhydrides, acyl imidazolide, acyl azides, or N-acyl benzotriazoles, followed by reaction with sodium borohydride to give the corresponding alcohols.
  • Chemical groups e.g., sugars such as glucose, can be added onto 5- hmC using a glycosy transferase such as an a-glucosyltransferase (AGT) or a BGT.
  • AGT a-glucosyltransferase
  • BGT a-glucosyltransferase
  • Useful glycosy transferases can accept a nucleobase in a nucleic acid as a substrate.
  • Exemplary BGT enzymes are found in bacteriophage, such as T4. The T4 BGT show little DNA sequence specificity, suggesting a
  • T4 BGT Variants of the T4 BGT can be used.
  • the structure of T4 BGT and the identities of key residues in the enzyme are well understood, facilitating the construction of forms of the protein incorporating one or more amino acid deletions or substitutions.
  • T4 BGT is a monomer comprising 351 amino acid residues and belongs to the ⁇ / ⁇ protein class. It is composed of two non-identical domains, both similar in topology to Rossmann nucleotide- binding folds, separated by a deep central cleft which forms the UDP-GIc binding site.
  • Amino acids participating in the interaction with UDP include Ile238 (interactions with N3 and 04 of the base); Glu272 (interactions with 02' and 03' of the ribose); Serl89 (interacting with Oi l of the a-phosphate); Argl91 (interacting with 012 of the a-phosphate); Arg269 (interacting with 06 of the ⁇ -phosphate and 022 of the ⁇ -phosphate); and Argl95
  • a variant T4 BGT can be used to add a sugar to a nucleic acid.
  • Variants optionally include an amino acid sequence at least 70% (e.g. at least 75%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to amino acids 1-351, 10-272 or 22-272 of T4 BGT.
  • assays for glycosylated nucleic acids e.g. changes in susceptibility to cleavage by a glycosylation-sensitive endonuclease
  • REBASE® www.neb.com, New England Biolabs, Ipswich, MA
  • the endonuclease preferentially binds to a hydroxymethylated cytosine or a glucosyl-oxy-methylated cytosine and cleave the bound nucleic acid at a defined distance from the recognition site.
  • Exemplary endonucleases include those whose amino acid sequences are identical to, or are at least 95% identical to, an enzyme selected from the group consisting of PvuRtslI, PpeHI, EsaSS310P, EsaRBORFBP, PatTI, Ykrl, EsaNI, SpeAI, BbiDI,
  • PfrCORFlI80P PcoORF314P, BmeDI, AbaSI, AbaCI, AbaAI, AbaUMB30RFAP and Asp60RFAP, as described in US Patent Application Publication No.
  • Example 1 Expression of mYOXl.
  • Example 2 Determination of activity of mYOXl.
  • methylation sites underlined is: 5'- ATTACACGCGCGATATCGTTAACGATAATTCGCGCGATTACGATCGATAACGCGTT AATA-3' (SEQ ID NO: 10).
  • the cytosine complementary to the subsequent guanine residue is also methylated, yielding a total of 24 methylated cytosines per double stranded DNA.
  • the assay mix contained in a final volume of 20 ⁇ _: 50 mM Bis-TRIS pH 6.0, 50 mM NaCI, 1 mM dithiothreitol (DTT), 2 mM ascorbic acid, 2 mM a-ketoglutarate, 100 ⁇ ferrous sulfate (FeSO 4 ), 2 ⁇ oligonucleotide (24x), and 4 ⁇ mYOXl.
  • the reaction mixture was incubated for 1 hour at 34°C.
  • the protein was digested using proteinase K (NEB) at a final concentration of 1 ⁇ g/ ⁇ L for 1 hour at 50°C.
  • the DNA was recovered by using QIAquick ® Nucleotide
  • the recovered DNA was digested by a mixture of 0.5 U nuclease PI (Sigma-Aldrich, St. Louis, MO), 5 U antarctic phosphatase (NEB), 2 U DNAse I (NEB) in 20 ⁇ _ total volume for 1 hour at 37°C.
  • the digested DNA was then subjected to LC-MS analysis.
  • LC-MS was done on Agilent 1200 series (G1316A UV Detector, 6120 Mass Detector, Agilent, Santa Clara, CA) with Waters Atlantis T3 (4.6 x 150 mm, 3 ⁇ , Waters, Milford, MA) column with in-line filter and guard.
  • mYOXl was active at pH 8.0, oxidizing a portion of the 5-mC to 5-hmC and 5-fC.
  • the enzyme was even more active at lower pH. For example, at pH 7.5, approximately 90% of the 5-mC residues were oxidized, with most of the product present as 5-hmC and 5-fC.
  • pH 7.3 the proportions of 5-mC and 5-hmC decreased, with increasing proportions of 5-fC and 5-caC.
  • mYOXl The activity of mYOXl was tested on single-stranded DNA (ssDNA) substrates and compared to that of a double-stranded DNA (dsDNA) with the same sequence under the same experimental conditions discussed for 24x oligo. Surprisingly, it was found that mYOXl oxidizes 5-mC in ssDNA as efficiently as dsDNA. Substrates included double-stranded "oligo 9"; "hemi- oligo 9,” a double stranded DNA identical to oligo 9 but lacking
  • RNA substrate having all its cytosines in 5-mC form: gggtctagaaataattttgtttaactttaagaaggagatatacatatgaaaatcgaagaaggtaaggtcaccatcac catcaccacggatccatggaagacgccaaaacataaagaaaggcccggcgccattctatcctctagaggatggaacc gctggagagcaactgcataaggctatgaagagatacgccctggttcctggaacaattgctttttacagatgcacatatc gaggtgaacatcacgtacgcggaatacttcgaaatgtccgttcggttggcagaagctatgaaacgatatgggctgaat acaatcacagaatcgtcgtatgtcgtttggcagaag
  • the assay conditions were as follows: 50 mM Bis-TRIS pH 6.0, 50 mM NaCI, 1 mM DTT, 2 mM ascorbic acid, 2 mM a-ketoglutarate, 100 ⁇ FeSO 4 , 1 g 5- mC RNA, and 4 ⁇ mYOXl.
  • the reaction mixture was incubated for 1 hour at 34°C.
  • the protein was digested using proteinase K (NEB) at a final concentration of 1 ⁇ g/ ⁇ L for 1 hour at 37°C.
  • the RNA was recovered by using QIAquick ® Nucleotide Removal Kit (QIAGEN, Valencia, CA). The recovered RNA was digested into nucleosides and analyzed by LC-MS as described in example 2A. The results were as follows:
  • the reaction mixture was incubated for 1 hour at 34°C.
  • the reaction mixture was then digested with proteinase K for 1 hour at 50°C.
  • the DNA was recovered by using QIAquick ® PCR Purification Kit (QIAGEN, Valencia, CA).
  • the recovered DNA was digested and analyzed by LC-MS as described in Example 2A. As shown, mYOXl efficiently oxygenates 5-mC in plasmid and genomic DNA samples.
  • Example 3 mYOXl can be used in conjunction with BGT.
  • An mYOXl/T4-BGT coupled assay was performed as described in Example 2A for genomic DNA (IMR90), with the following exceptions: 50mM Hepes pH 7.0 was used instead of Bis-Tris pH 6.0, and 40 ⁇ uridine diphosphoglucose (UDP-GIc) and 50 U T4 BGT were added in the oxidation reaction.
  • 50mM Hepes pH 7.0 was used instead of Bis-Tris pH 6.0
  • 40 ⁇ uridine diphosphoglucose (UDP-GIc) and 50 U T4 BGT were added in the oxidation reaction.
  • bacterial genomic DNA for bacterial genomic DNA (MG1655), the reaction was carried out exactly as described in Example 2A. Then the reaction mixture was digested with proteinase K for 1 hour at 50°C. The sample was then treated with lOOmM NaBH 4 , 40 ⁇ uridine diphosphoglucose (UDP-GIc) and 50 U T4-BGT in lx NEBuffer 4 (NEB) and incubated for 1 hour at 37°C. The DNA was recovered by using QIAquick ® PCR Purification Kit (QIAGEN,
  • MG1655 oxidation/ + 29.3% - 3.0% 67.7% - reduction
  • the effects of increasing ATP concentration on the activity of mYOXl when coupled with the activity of T4-BGT in the presence of NaBH 4 and UDP- Glc were tested. ATP concentrations higher than 1 mM exhibit inhibiting effects on the activity of mYOXl to convert 5-mC to 5-hmC.
  • the reaction was carried out exactly as described in Example 2A for oligo 9 except for the duration of the oxidation reaction (20 minutes instead of 1 hour), and the presence of varying amounts of ATP.
  • the reaction mixture was then digested with proteinase K and glucosylated using T4 BGT as described above for MG1655 genomic DNA. The DNA was recovered by using
  • Example 4 Qualitative and quantitative assays for characterization of the mYOX family of enzymes.
  • Immunodot-blot assay This is a qualitative, but relatively fast assay. Many samples can be tested simultaneously, which can be used for
  • LC-MS analysis To quantify mYOXl oxidation products, LC-MS analysis was performed on a reverse-phase Waters Atlantis T3 C18 column (3 ⁇ , 4.6 x 150 mm) with an Agilent 1200 LC-MS system equipped with an Agilent G1315D DAD detector and an Agilent 6120 Quadruple MS detector. A binary solvent system with ammonium acetate (10 mM, pH 4.5) and
  • each nucleoside peak was quantified at its absorption maximum and adjusted by the extinction coefficient constant.
  • Example 5 5-hmC specific endonuclease assay.
  • C m CGG methylase
  • Example 6 Methods for sequencing the methylome
  • Bioinformatic analysis of the sequencing reads utilized the PI ends to mark the enzyme's cleavage sites. After mapping the read back to the reference genome, the modified cytosine was determined to be located at a fixed distance away from the cleavage sites and on either side.

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Abstract

L'invention concerne des 5-méthylpyrimidine oxygénases et leur utilisation dans la modification d'acides nucléiques.
PCT/US2013/068298 2012-11-06 2013-11-04 Compositions et procédés pour l'oxygénation d'acides nucléiques contenant de la 5-méthylpyrimidine Ceased WO2014074453A1 (fr)

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US13/827,087 US9040239B2 (en) 2012-03-15 2013-03-14 Composition and methods of oxygenation of nucleic acids containing 5-methylpyrimidine
US13/826,395 2013-03-14
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US13/826,395 US9267117B2 (en) 2012-03-15 2013-03-14 Mapping cytosine modifications
US13/827,885 US9121061B2 (en) 2012-03-15 2013-03-14 Methods and compositions for discrimination between cytosine and modifications thereof and for methylome analysis
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